Radiotherapy is frequently used alone or in combination with chemotherapy or surgery in the treatment of head and neck cancer. Nearly all patients receiving regional radiotherapy with curative intent develop oropharyngeal complications. The damage to normal tissues that fall within the portals of radiation result in a number of side- effects that include oral mucositis, ulcerations, salivary hypofunction, dysphagia, infections, and dental decay. The debilitating sequelae of radiotherapy impact the patient's quality of life and negatively affect the continuation of cancer therapy. Current management of these complications is largely palliative, and prevention of radiation toxicity to normal tissues provides the best treatment prospect to reduce the impact on patient's well-being. Despite improvements in radiation delivery techniques, radiation-induced toxicity continues to be a limiting factor in cancer treatment. With few radioprotectors in clinical use, novel interventions are being explored to prevent or treat radiation injury. We have demonstrated that increased expression of Toulsed-like kinase 1B (TLK1B) protects normal salivary cells against radiation damage in vitro, and the direct transfer of cell-permeable TAT-TLK1B protein in salivary glands in vivo effectively mitigates irradiation-induced salivary hypofunction. But, similar to most radioprotectors aimed at protecting normal tissues, TLK1B carries the unavoidable risk of protecting tumors and limiting the efficacy of cancer treatment. Therefore, to reduce morbidity without compromising cancer curability, the central goal of the proposal is to develop TLK1B as a radioprotector for normal cells, but a radiosensitizer for tumors. Degradation of the extracellular matrix by proteases including matrix metalloproteinases (MMPs) is a hallmark of tumor growth, invasion, and metastasis. Elevated expression of proteolytically active MMPs, especially MT1 (membrane-type 1)-MMP, MMP-2, and MMP-9, is associated with a variety of cancers including head and neck cancer (HNSCC). Capitalizing on the proteolytic enzyme-rich milieu around cancer cells, we will engineer TAT-TLK1B with MT1-MMP cleavage motif and validate recombinant protein transduction and efficacy in head and neck squamous cell carcinoma (HNSCC) cell lines in vitro (Aim 1). The functionality of our approach will be tested in vivo in a nude mouse HNSCC xenograft model (Aim 2). We recently obtained encouraging preliminary data showing significantly reduced transduction of MT1-MMP- susceptible TAT-TLK1B in cultured HNSCC cells. We have confidence that our proposed protein engineering approach has the potential to effectively discriminate against protein entry in cancer cells. The cleavage of MT1-MMP-sensitive TAT-TLK1B protein by active proteases in a tumor milieu in vivo will transduce a kinase-deficient, dominant-negative protein in tumor cells that could sensitize them to ionizing radiation. The development of TLK1B as a radioprotector that enhances the therapeutic index not only by selective protection of normal tissue, but also by sensitization of tumor tissue will greatly augment its potential for clinical use.